Cellular thiol plays an important role in a biological system. A change in thiol concentration is associated with oxidation stress involved in poison and disease. Glutathione is the most abundant cellular thiol. It acts as an antioxidant on cells, so that it protects the cells from many cancerogenic substances. Homocysteine is a risk factor for diseases such as cardiovascular disease and Alzheimer's disease. A thiol group in a cysteine residue is involved in the three-dimensional structure of a protein through the formation of a disulfide bond. The absence of cysteine may cause serious health problems. Accordingly, the detection of intracellular thiol is extremely important for examining the functions of cells.
Ellman reagent is a thiol-detecting reagent, which has been broadly used from of old (Non-Patent Document 1). However, since an assay using this reagent is based on a change in absorbance, the reagent can be used only in vitro. Several methods for detecting thiol, involving a sensitive fluorescent probe, have been reported. A fluorescein derivative protected by 2,4-dinitrobenzenesulfonyl ester has been reported (Non-Patent Documents 2 and 3). This probe reacts with a biological thiol to generate a high fluorescence intensity in a short reaction time. However, this probe is problematic in that since its sulfonyl ester is hydrolyzed in an aqueous solution, background fluorescence may be generated.
Recently, a fluorescent probe having a 2,4-dinitrobenzenesulfonamide (DNB) group has been reported (Non-Patent Documents 4 and 5). A sulfonamide group is resistant to hydrolysis, and thus, no background fluorescence is generated.    Non-Patent Document 1: Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.    Non-Patent Document 2: Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem. Int. Ed. 2005, 44, 2922.    Non-Patent Document 3: Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T. Angew. Chem. Int. Ed. 2006, 45, 1810.    Non-Patent Document 4: Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem. Int. Ed. 2007, 46, 8445.    Non-Patent Document 5: Bouffard, J.; Kim, Y.; Swager, T. M.; Weissleder, R.; Hilderbrand, S. A. Org. Lett. 2008, 10, 37.